Tive latency type I cells. EBV reactivation by BCR PD-148515 site signaling (anti-IgM
Tive latency type I cells. EBV reactivation by BCR signaling (anti-IgM), protein kinase C activation [1] and TGF- signaling provide unique models to investigate ATO-mediated EBV protein degradation. First, a combination of ATO with each of these signaling reagents PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/27486068 was applied to cells, and the proteasome inhibitor MG132 was added prior to harvest. As shown in Fig. 5e, ATO inhibited anti-IgM-, PMA- and TGF–mediated EBV reactivation. The expression of Zta, Rta and BMRF1 were activated by anti-IgM, PMA or TGF-, and inhibited after cotreatment with ATO. Importantly, MG132 rescued theATO-mediated inhibition of EBV reactivation induced by anti-IgM, PMA or TGF- (Fig. 5e). Therefore, ATO disrupts the EBV infection cycle and inhibits EBV gene expression through activation of global cellular protein ubiquitination. These results indicate that ATO induces the rapid sumoylation of Zta, Rta and BMRF1, resulting in their ubiquitination and proteasome-dependent degradation. Thus, ATO-mediated EBV protein degradation is dependent on SUMO-regulated protein ubiquitination and proteasomemediated degradation.Discussion We have shown that ATO inhibits EBV reactivation through ubiquitin-mediated degradation. The consequences of this were inhibition of EBV replication and induction of cell death in EBV-positive cells. This result is consistent with a previous report that loss of the EBV genome and lytic gene expression leads to the loss of the malignant phenotype and cell viability in EBV-positive Burkitt’s lymphoma cells [65?7]. EBV lytic genes are expressed in 29 of lymphoma patients according to Dr. Liu’s report [67]. In EBV-positive cells, the lytic viral proteins regulate diverse homeostatic cellular functions including inflammation and angiogenesis. Thus, the small portion of cells in the lytic cycle may support tumor cell growth and survival by providing cell growth factors and other signals. Diminishing lytic gene expression in cellsYin et al. Virology Journal (2017) 14:Page 8 ofFig. 5 EBV lytic proteins were ubiquitined and sumoylated in response to ATO treatment and this effect was rescued using proteasome and SUMO inhibitors in EBV latency type I Mutu cells. a 1 nM of ATO induced SUMO1 expression. b Co-IP with antibodies against Zta and western blotting using antibodies against ubiquitin. c MG132 at various concentrations (0.1 M ?5 M for 16 h) rescued EBV spontaneous reactivation that was reduced in response to 1 nM of ATO. d Proteasome (MG132) SUMO1 (Ginkgolic Acid, GA) inhibitors rescued the reduction in EBV reactivation in response to ATO. Cells were treated with 1 nM of ATO for 3 days and with/without MG132 or GA at indicated concentrations (M) for 4 h. e 1 nM ATO inhibited the anti-IgM-, PMA-, and TGF–induced EBV reactivation and MG132 (1 M for 16 h) abrogated this effectexposed to ATO eradicates EBV genome replication and results EBV-dependent cell death. Spontaneous EBV reactivation provides us a cell system to evaluate the anti-tumor effect of ATO on lymphoma cells. The expression of EBV lytic genes is decreased significantly in response to ATO treatment, suggesting that ATO promotes EBV lytic protein degradation (Fig. 1). Further, the growth rate in cell populations with spontaneous EBV reactivation is faster than in cells treated with ATO. More importantly, inhibiting EBV lytic gene expression in cells exposed to ATO impedes the proliferation of these cells (Figs. 3 4), but this was not observed in EBV-negative cells. In EBV-associ.
